Pyrite shrink-film laminate as a hydroxyl radical generator

ABSTRACT

Disclosed are hydroxyl radial generating devices, comprising: a substrate layer; and a pyrite layer configured to produce hydroxyl radicals. Another aspect relates to a method for producing a hydroxyl radical generating device, comprising: providing a polymeric substrate layer; placing a layer of pyrite on a surface of the polymeric substrate layer to form a multi-layer structure; and applying heat to the multi-layer structure such that at least the surface of the polymeric substrate layer contracts; wherein the layer of pyrite contracts to a lesser extent than the surface of the polymeric substrate layer providing a textured surface comprising the pyrite layer. Also disclosed is a method of analysis, comprising: placing a solution comprising a biological substance on a sample site of a hydroxyl generating device comprising a surface of pyrite; incubating the solution; and analyzing a sample including proteolytic fragments of the biological substance.

STATEMENT REGARDING FEDERALLY SPONSORED R&D

This project was supported by NSF-IDBR 0852796, the Bio-molecularInteraction Technologies Center, a NSF Industry/University CooperativeResearch Center, NIH DP2 OD007283-01 and the U.S. DOE DE-EE0005324,funded by the SunShot Next Generation Photovoltaics II (NextGen PVII)program.

INCORPORATION BY REFERENCE TO ANY PRIORITY APPLICATIONS

Any and all applications for which a foreign or domestic priority claimis identified in the Application Data Sheet as filed with the presentapplication are hereby incorporated by reference under 37 CFR 1.57.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This application is directed at simple, easy to use, and low cost toolsand methods for the analysis of biologics for pharmaceutical and otheruses.

2. Description of the Related Art

Oxidative footprinting of proteins is typically carried out bysynchrotron or gamma source radiolysis of water or laser photolysis ofhydrogen peroxide. Both approaches require a sizable investment ininfrastructure.

SUMMARY OF THE INVENTION

The structure of proteins, DNA and RNA and their complexes oftendictates their biological function. In “footprinting”, the solventaccessibility of the residues that constitute proteins and nucleic acidsis determined from their reactivity to an exogenous reagent such as thehydroxyl radical (.OH). While .OH generation for protein footprintingcan be achieved by radiolysis, photolysis and electrochemistry, wepresent simpler, lower cost, yet more elegant and more practicalsolutions. In some embodiments, an iron-bearing inorganic or organicfilm capable of providing Fe²⁺ to react with H₂O₂, e.g., a thin film ofpyrite (cubic FeS₂) nanocrystals deposited on a solid surface, in someapproaches onto a shape memory polymer (e.g., a commodity shrink film ofthe type used in shrink-wrap applications) generates sufficient .OH viaFenton chemistry for oxidative footprinting analysis of biomolecules.The term shrink-wrap where used herein connotes the same or similarmaterials that are used in shrink-wrap applications, including shapememory polymers. We demonstrate that varying either time or H₂O₂concentration yields the required ·OH dose-oxidation responserelationship provided in footprinting. A simple and scalable samplehandling protocol is enabled by thermoforming the “pyrite shrink-filmlaminate” into a microtiter plate format. The low-cost and malleabilityof pyrite shrink-film laminate will facilitate its use in highthroughput screening applications as well as integration intomicrofluidic devices.

Some embodiments relate to a hydroxyl radial generating device,comprising: a substrate layer; and a pyrite layer configured to produce.OH.

In some embodiments, the substrate layer comprises a material configuredto shrink when heated by at least 50%, the pyrite layer configured toshrink when heated by a lesser amount such that when the substrate layerand the pyrite layer are shrunken the pyrite layer comprises a texturedsurface.

In some embodiments, the substrate layer and the pyrite layer are in ashrunken configuration.

In some embodiments, the hydroxyl radical generating device comprises atleast one well for retaining a liquid sample.

In some embodiments, the hydroxyl radical generating device comprises aplurality of wells for retaining a plurality of liquid samples.

In some embodiments, the hydroxyl radical generating device comprises amicrotiter plate comprising a plurality of sample wells, each of saidwells having the pyrite layer therein.

In some embodiments, the hydroxyl radical generating device comprises amicrofluidic device comprising at least one sample channel and a samplesite in fluid communication with the sample channel, the sample sitehaving the pyrite layer therein.

In some embodiments, the pyrite layer comprises pyrite crystals.

In some embodiments, the pyrite layer comprises nano-scale crystals.

In some embodiments, a shrunken configuration of the substrate layer hasan area within a periphery thereof at a surface thereof that is at least50% less than or even as much as 80% less than an area within theperiphery at the surface in the pre-shrunk configuration.

In some embodiments, a shrunken configuration of the substrate layer hasan area within a periphery thereof at a surface thereof that is at least95% less than an area within the periphery at the surface in thepre-shrunk configuration.

Some embodiments relate to a method for producing a hydroxyl radicalgenerating device, comprising: providing a polymeric substrate layer;placing a layer of pyrite on a surface of the polymeric substrate layerto form a multi-layer structure; and applying heat to the multi-layerstructure such that at least the surface of the polymeric substratelayer contracts; wherein the layer of pyrite contacts to a lesser extentthan the surface of the polymeric substrate layer providing a texturedsurface comprising the pyrite layer.

In some embodiments, the layer of pyrite comprise a layer of pyritenanocrystals.

In some embodiments, after placing the layer of pyrite on the surface,the layer of pyrite comprises a minimum thickness of between 20 and 80nm.

Some embodiments further comprise forming at least one sample site inthe multi-layer structure.

Some embodiments further comprise thermoforming a well by heating themulti-layered structure above the glass transition temperature of thepolymeric substrate layer and drawing the multi-layered structure into asample site form.

In some embodiments, the sample site form comprises a structurecomprising a plurality of openings and wherein drawing comprisesdeforming the multi-layer structure at each of the openings into theopenings.

In some embodiments, applying heat causes a shrinkage of at least about50% of the area of the surface.

In some embodiments, the layer of pyrite is deposited by asolution-based deposition method.

In some embodiments, the layer of pyrite is deposited by a gas-phasedeposition method.

Some embodiments relate to a method of analysis, comprising: placing asolution comprising a biological substance on a sample site of ahydroxyl generating device comprising a surface of pyrite; incubatingthe solution; and analyzing a sample including proteolytic fragments ofthe biological substance.

Some embodiments further comprise performing drop depositionfootprinting by placing the solution in a well of a microtiter plate andthereafter incubating and analyzing the sample.

Some embodiments further comprise placing the sample on a sample site ofa microfluidic device and thereafter incubating and analyzing thesample.

In some embodiments, the surface of pyrite comprises a textured surfacecomprising nanocrystals.

Some embodiments further comprise combining an amount of H₂O₂ with thesolution prior to incubating the solution.

Some embodiments further comprise incubating the solution by vibratingthe sample test device and the solution.

Some embodiments further comprise combining the solution with a reactionhalting medium after incubating.

Some embodiments further comprise activating a surface of a well priorto placing the solution therein.

In some embodiments, the biological substance includes DNA.

In some embodiments, the biological substance includes RNA, DNA, or oneor more proteins.

In some embodiments, the biological substance includes one or moreproteins.

Some embodiments relate to a kit for analyzing a sample, comprising: asubstrate comprising a layer of pyrite disposed thereon; and one or morereagents that catalyze production of hydroxyl radicals from the layer ofpyrite.

In some embodiments, the one or more reagents catalyze a Fenton chemicalreaction.

In some embodiments, the one or more reagents comprise solution(s) ofhydrogen peroxide (H₂O₂) and/or ascorbate.

In some embodiments, the substrate and pyrite layer comprise a portionof a microtiter plate.

In some embodiments, the substrate and pyrite layer comprise a portionof a microfluidic device.

In some embodiments, crystals are disposed, e.g., deposited, on a layer.A concentrating process such as heat-shrinking is used to create highsurface area substrates for improved chemical reactions. The crystalscan be nano-scale crystals in one example. Although pyrite is discussedin depth herein, the crystals can be other iron (e.g., iron oxide) ornon-iron based compounds in certain embodiments.

In one embodiment, pyrite nanocrystals are deposited on shrink-filmplastic. Heat is applied to the plastic to shrink the plastic. Theresult is a highly textured laminate surface. Wells are thermoformedinto the surface. The wells can retain sample drops. The iron in thepyrite catalyzes the production of hydroxyl radicals by an efficientprocess, such as via Fenton chemistry.

This application describes an apparatus that is of great interest to thepharmaceutical industry. In one embodiment, the apparatus includes apyrite shrink-film laminate. The pyrite shrink-film laminate can be usedto generate hydroxyl radical for oxidative footprinting. Anotherapparatus is a bench top generator for producing hydroxyl radicals. Thegenerator can be part of a kit that may include a pipette or other smallvolume dispenser.

In one embodiment a method is provided for producing an oxidativefootprinting device. In the method, a polymeric substrate layer isprovided. A layer of an iron-bearing inorganic or organic film capableof providing Fe²⁺ to react with H₂O₂, e.g., pyrite, is placed on asurface of the polymeric substrate layer to form a multi-layerstructure. Heat is applied to the multi-layer structure such that atleast the surface of the polymeric substrate layer contracts. The layerof pyrite (or other inorganic or organic film capable of providing Fe²⁺to react with H₂O₂) contracts to a lesser extent than the surface of thepolymeric substrate layer. This difference in contraction results in thepyrite layer buckling, providing a highly textured surface comprisingthe pyrite layer.

In another embodiment, a drop deposition footprinting method isprovided. In the method, a solution comprising a biological substance isplaced on a sample site of a sample test device comprising at least onewell surrounded by a highly textured surface of pyrite (or otherinorganic or organic film capable of providing Fe²⁺ to react with H₂O₂).The solution is incubated. A sample including proteolytic fragments ofthe biological substance is analyzed.

In another embodiment, a hydroxyl radical generator is provided thatincludes a first layer and a second layer. The first layer comprises amaterial at a surface of the first layer. The surface is surrounded by aperiphery. The first layer has pre-shrunk configuration and a shrunkenconfiguration. The shrunken configuration has an area within theperiphery at the surface that is less than, e.g., a least 50% less than,the area within the periphery at the surface in the pre-shrunkconfiguration. The second layer comprises a material configured toproduce .OH in the presence of a biological sample. The second layer isconfigured to shrink to a lesser extent than the surface of the firstlayer within the periphery, such that the second layer is highlytextured when the first layer is in the shrunken configuration.

The second layer in the foregoing embodiment can include an inorganic ororganic film capable of providing Fe²⁺ to react with H₂O₂, e.g., formedusing pyrite. The pyrite can be in a nanocrystalline form.

In another embodiment, a microtiter plate is provided. The microtiterplate has a plurality of sample wells. At least one of the wells has alayer of pyrite therein. In other embodiments, the well or wells caninclude any inorganic or organic film capable of providing Fe²⁺ to reactwith H₂O_(2.)

In another embodiment, a microfluidic device is provided. Themicrofluidic device comprises at least one sample channel and a samplesite. The sample site is in fluid communication with the sample channel.The sample site can have a layer of pyrite therein. In otherembodiments, the sample site can include any inorganic or organic filmcapable of providing Fe²⁺ to react with H₂O_(2.)

In another embodiment, a kit is provided for making a bench topgenerator for producing hydroxyl radicals for molecular footprinting.The kit includes a shrinkable polymeric layer and a vial of anyinorganic or organic substance capable of providing Fe²⁺ to react withH₂O₂. The kit also includes a template for forming fluid channels orfluid wells.

The apparatuses and methods herein can be configured as diagnostic andresearch and development tools.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects and advantages are described belowwith reference to the drawings, which are intended to illustrate but notto limit the inventions. In the drawings, like reference charactersdenote corresponding features consistently throughout similarembodiments. The following is a brief description of each of thedrawings.

FIG. 1. (A) illustrates one method for fabricating an embodiment of apyrite shrink-film laminate. (B) includes photographs of a substratebefore (left) and after (right) thermal shrinking (C) shows an image ofa substrate modified to include microwells. (D) shows a schematic of theprocess for thermoforming the laminate to form microwells.

FIG. 2 (A) shows an example embodiment in which a microtiter plate isprovided that includes an array of wells formed therein for testingsamples. (B) illustrates a process in which the production of sampletest devices can be scaled by providing for production more than onesuch device at the same time.

FIGS. 3 (A) and (B) are top down SEM images showing thehigh-surface-area pyrite micro-structure of laminate at medium and highresolution. (C) is a SEM image showing the cross-section of a pyritenanocrystal film airbrushed onto silicon in a single pass. Although itis not used in some embodiments of the sample apparatus, the siliconsubstrate facilitates fracturing for cross sectional imaging of thepyrite nanocrystal film. (D) shows relative fluorescence of solutiondrops of 3 μL containing fluorescent dye, 2 mM ascorbate and 8 mM H₂O₂,which were incubated for 1 min in a microfuge tube (a), in a pyriteshrink-film microwell (b) and a pyrite microwell with vibration (c).

FIG. 4 illustrates a process flow summary for drop deposition oxidativefootprinting, including some or all of the following stages: i) surfaceoxidation is removed by mild acid followed by pH neutralization withmultiple water washes; ii) H₂O₂ and ascorbate are added to a proteinsample in standard reaction buffer to the desired concentrations; iii)the reaction mixture is pipetted into microwell and incubated withvibration for a defined period of time; iv) the sample is transferred tothe quench solution; v) aliquots are removed from the quenched samplefor proteolytic fragmentation and mass spectral analysis. In variantsthat are not illustrated, multiple samples can be processed in parallelusing manual or robotic multichannel pipettes with or without acid wash.

FIG. 5 shows relative .OH production assayed and quantitated byfluorescent dye degradation. (A) Quantification of .OH production bypyrite shrink-film laminate as a function of H₂O₂ and ascorbateconcentration at constant 5 min incubation; (B) Three μL bufferedsolution drops containing dye and 8 mM H₂O₂ and 2 mM ascorbate wereincubated on three different samples: a mineral pyrite surface, a pyritenanocrystal film airbrushed onto a silicon wafer, and the pyriteshrink-film laminate. The samples were vibrated to aid mixing. Thecontrol samples contained the same solutes but were not incubated onpyrite; (C) Dose-response curves relating .OH generation by pyriteshrink-film laminate as a function of incubation time (solid line) andH₂O₂ concentration (dashed line) with 10 mM H₂O₂ and 2 mM ascorbatepresent in solution at a constant incubation time of 5 min.

FIG. 6 shows a comparison of the relative oxidation by 2 μMFe(II)-EDTA-[Fe(EDTA)]²⁻—(roughly the amount of ferrous iron releasedinto solution from pyrite shrink-film laminate) and the laminate itself.Two mM ascorbate with 15 mM H₂O₂ was present in both reactions thatincubated for 1 min. Relative production of .OH was assayed bydegradation of a fluorescent dye.

FIG. 7 shows oxidation of the protein Programmed Death-1 (PD-1) bypyrite shrink laminate. Three μL of PD-1 in buffer containing 1 mMascorbate and the indicated H₂O₂ concentration were incubated for 1 minwith vibration on pyrite shrink-film laminate. Aliquots of the proteinwere proteolyzed and prepared for mass spectral analysis. Panel (A)provides a MALDI-MS1 analysis of the N-terminal tryptic peptide(residues 1-36) of PD-1 showing the multiples of +16 mass increasesdetected as a function of increasing concentrations of H₂O₂. Panel (B)provides the fraction of unmodified peptide as a function of H₂O₂concentration. The solid line is an exponential fit to the data. Theinsert is a ribbon representation of the PD-1 structure highlighting theN-terminal peptide in green and the residues Y4, W8, W26, and M31 thatare highly susceptible to oxidation.

FIG. 8 shows the .OH production from pyrite shrink-film laminate as afunction of storage time in a closed container at room temperature inthe dark. Ambient humidity was quantified by the dye degradation assayconducted at constant experimental conditions. The data show that pyriteshrink-film laminate retains full activity when stored over the 14months assayed. The data shown are normalized the first data point.

FIG. 9. Measurement of the amount of iron released from pyriteshrink-film laminate during production of .OH.

FIG. 10. Contact with pyrite shrink laminate does not cause denaturationof PD-1. Drops containing PD-1 in standard reaction buffer wereincubated on the surfaces for 25 min with vibration prior to analysis ofthe protein's global conformation. (A) Intrinsic tryptophan fluorescencewas used to determine whether exposure of the protein PD-1 (□) to barepolyolefin (O) or pyrite shrink laminate (Δ) unfolds the protein. PD-1denatured in 2 M urea (shaded ♦) is shown for comparison. (B)Sedimentation velocity analytical ultracentrifugation analysis measuresthe global conformation of biological macromolecules. The nativeconformation of PD-1 is maintained following exposure of the protein tobare polyolefin or pyrite shrink laminate is the invariance of themeasured sedimentation (S) and diffusion coefficients (D). S and D arecorrected to standard conditions (S20,w and D20,w) to account fortemperature (20° C.) and buffer properties within the experiment.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

While the present description sets forth specific details of variousembodiments, it will be appreciated that the description is illustrativeonly and should not be construed in any way as limiting. Furthermore,various applications of such embodiments and modifications thereto,which may occur to those who are skilled in the art, are alsoencompassed by the general concepts described herein. Each and everyfeature described herein, and each and every combination of two or moreof such features, is included within the scope of the present inventionprovided that the features included in such a combination are notmutually inconsistent.

Introduction

In “footprinting,” solvent accessibility of individual residues ofbiological macromolecules is measured by their reactivity to anexogenous reagent (V. Petri and M. Brenowitz 1997 Current opinion inbiotechnology 8: 36-44; K. Takamoto and M. R. Chance 2006 Annual reviewof biophysics and biomolecular structure 35: 251-276). The hydroxylradical (.OH) is an effective footprinting probe due to its highreactivity and small size (G. V. Buxton 1988 J Phys Chem Ref Data 17:513-886). Nucleic acid .OH footprinting monitors backbone polysaccharidecleavage (T. D. Tullius and B. A. Dombroski, 1986 Proceedings of theNational Academy of Sciences of the United States of America 83:5469-5473). Mapping of DNA structure and protein binding (T. D. Tulliusand B. A. Dombroski, 1986 Proceedings of the National Academy ofSciences of the United States of America 83: 5469-5473) was followed bystudy of RNA structure and folding (B. Sclavi et al. 1998 Science 279:1940-1943). Quantification of side chain oxidation is the dominant modeof protein footprinting analyses (K. Takamoto and M. R. Chance 2006Annual review of biophysics and biomolecular structure 35: 251-276).Footprinting isotherms and time progress curves linked to localstructural transitions resolve macromolecular binding and foldingmechanisms (M. Brenowitz et al. 2002 Current opinion in structuralbiology 12: 648-653).

Ascorbate-driven Fenton chemistry mediated by [Fe(edta)]²⁻ is a widelyused .OH footprinting method (T. D. Tullius and B. A. Dombroski, 1986Proceedings of the National Academy of Sciences of the United States ofAmerica 83: 5469-5473). The coupled reactions are:

Fe²⁺+H₂O₂→Fe³⁺+HO.+OH⁻  (1); and

Fe³⁺+H₂O₂→Fe²⁺+HOO.+H⁺  (2)

Variations include using reaction 1 alone to study fast structuraltransitions (I. Shcherbakova, S. et al. 2006 Nucleic acids research 34:e48), using dissolved O₂ instead of H₂O₂ (K. Takamoto et al. 2004Journal of molecular biology 343: 1195-1206), and substitutingperoxonitrous acid for [Fe(edta)]²⁻ and H₂O₂ (P. A. King et al. 1993Nucleic acids research 21: 2473-2478). Radiolysis of water using lowflux gamma (J. J. Hayes et al. 1990 Methods in enzymology 186: 545-549)and high flux synchrotron radiation (B. Sclavi et al. 1997 Journal ofmolecular biology 266: 144-159; S. D. Maleknia et al. 1999 Analyticalchemis-try 71: 3965-3973), a pulsed electron beam (C. Watson et al. 2009Analytical chemistry 81: 2496-2505), laser photolysis of H₂O₂ (D. M.Hambly and M. L. Gross 2005 Journal of the American Society for MassSpectrometry 16: 2057-2063) and electrochemical oxidation (E. B. Monroeand M. L. Heien 2013 Analytical chemistry 85: 6185-6189) are used togenerate .OH for footprinting. The latter approaches require specializedequipment for radical generation, management of fluid flow and samplecollection.

The mineral iron pyrite (cubic FeS₂) supports Fenton chemistry (C. A.Cohn et al. 2004 Abstr Pap Am Chem S 228: U698-U698). Powdered pyrite ina microfluidic device footprints DNA and RNA (J. C. Schlatterer and M.Brenowitz 2009 Methods 49: 142-147; J. C. Schlatterer et al. 2012Biochemical and biophysical research communications 425: 374-378).However, powdered mineral proved incompatible with multiplexedmicrofluidic mixers (Jones, C D, Schlatterer, J C, Brenowitz, M &Pollack L; unpublished). Herein we describe the deposition of pyritenanocrystals (J. Puthussery et al. 2011 Journal of the American ChemicalSociety 133: 716-719) onto shape memory polymer (e.g., commodity shrinkfilm or a layer of film; D. Nguyen et al. 2010 Lab on a chip 10:1623-1626) to create a novel mediator of Fenton chemistry. Thermallyinducing the pyrite coated shrink film to retract causes the stiffernanocrystalline layer to buckle resulting in a highly reactive, wrinkledand robustly integrated laminate of pyrite nanocrystals. Sample wellscan be thermoformed into the laminate in a standard well format thatenables footprinting studies to be carried out by the simple depositionand removal of sample drops. We demonstrate the utility of pyriteshrink-warp laminate for the controlled generation of .OH for theoxidative protein footprinting.

Materials and Methods Fabrication and Characterization of PyriteShrink-Film Laminate

Pyrite nanocrystals can be prepared via a hot-injection method based ona published synthesis (J. Puthussery et al. 2011 Journal of the AmericanChemical Society 133: 716-719) as detailed in the Examples. The pyritecolloidal suspension can be spray coated onto 4″×4″ shape-memorypolyolefin sheets (Cryovac D955, Sealed Air) using a handheld BadgerModel 200 airbrush (FIG. 1A). Polyolefin sheets can bepinned to PMMAsubstrates and mounted vertically in a fume hood. Spray coating isperformed twice at room temperature with manual sweeps of ˜3 s from 8″descending from top to bottom; 5 mL of suspension are consumed for eachcoat. Ligand exchange is not performed.

The coated polyolefin sheets are shrunk at 160° C. by waving a heat gun(HL 1810 S, Steinel) above its surface (FIG. 1A). The polyolefin quicklyshrinks, e.g., by 95% (FIG. 1B; D. Nguyen et al. 2011 Biomicrofluidics5: 22209). A weight such as one or more binder clips is attached to thecorners to prevent the sheets from folding over. Microwells arethermoformed into the laminate (FIGS. 1, C & D) using a templatepatterned into 3 mm thick PMMA (McMaster-Carr) to 384 well platespecifications with a CO₂ laser cutter (Versalaser). Pyrite shrink-filmlaminate is placed on top of the template, a vacuum is applied and thelaminate gently heated above the glass transition temperature ofpolyolefin (125° C.) with the heat gun. The negative pressure pulls thelaminate into the template vias to form the microwells (FIG. 1D).

FIG. 2A shows a microtiter plate that can be produced in the processillustrated in FIGS. 1(a)-1(d). The microtiter plate can allow efficienttesting in a plurality of wells formed in the plate. FIG. 2B illustratesthat a scalable process can be used to produce many sample test devicesat the same time. In one approach a forming plate is provided. Theforming plate can have as many windows as sample test devices to beproduced. In this case, there are twelve windows. But in variants therecan be more than twelve, including more than 20, more than 50, more than100 or more than 500 windows. A structure produced by the processillustrated in at least the left four images of FIG. 1(a) and FIG. 1(b)can be used to produce a plurality of concentrated surface layerstructure, e.g., a concentrated laminate structures comprising awrinkled pyrite surface. Each concentrated surface layer structure canbe placed in a corresponding window and subject to negative pressurethrough a well or channel-forming template. The result can be pluralityof sample test devices with wells or channels of desired depth or shape.

To measure Fe²⁺ release from pyrite shrink-film laminate, 3 μL drops ofsolution containing 1 mM ascorbate and H₂O₂ at the indicatedconcentration are pipetted into wells and incubated with vibration for60 s. The sample is collected and stock solutions of sodium acetate,hydroxylamine-HCl and 1,10′-phenanthroline are added to finalconcentrations of 120, 6, and 500 mM, respectively. The final volume of1.2 mL is incubated, e.g., for 10 min at room temperature. The 508 nmabsorption of the Fe²⁺—1,10′-phenanthroline chelate is compared to acalibration curve constructed from known concentrations of ferrousammonium sulfate hexahydrate (Sigma).

Drop Deposition Oxidation by Pyrite Shrink-Film Laminate

A 16 well pyrite shrink laminate chip (FIG. 1C) is affixed to atabletop-mounted 6 V, 10,000 RPM vibration motor (Amico UPC610-696811493, Amazon.com). Surface oxidation is removed by adding 5 μLof 0.1 M HCl to microwells for 5 min followed by several water washes toneutralize the acid (FIG. 3). Measurement of the fluorescence loss of anaromatic dye is a convenient way to assess relative rates of .OHproduction (Example 4)(I. Shcherbakova, S. et al. 2006 Nucleic acidsresearch 34: e48; B. Ou et al. 2002 Journal of agricultural and foodchemistry 50: 2772-2777; and F. Chen et al. 2002 J. Phys. Chem. 106:9485-9490).

In our protein studies, PD-1 is diluted to 100 μM in standard reactionbuffer prior to oxidation experiments. Aliquots of recombinant BirAtagged mouse Programmed Death 1 (PD-1) is dialyzed into standardreaction buffer (20 mM Sodium Cacodylate pH 8.0, 50 mM NaCl, 1 mM EDTA);assays to test the retention of the protein's native fold are describedin Example 10.

FIG. 4 illustrates one method of ‘drop deposition oxidation’. Just priorto initiating oxidation, 0.6 and 2.4 μL of stock solutions of sodiumascorbate and H₂O₂, respectively are added to 27 μL of protein or dye inbuffer to the desired concentrations. A 3 μL aliquot is pipetted into apyrite shrink-film laminate microwell, vibration is provided (e.g., for1 min), vibration is terminated and the sample pipetted to 27 μL of a‘quench solution’ containing 34 μM thiourea, 12 μM methioninamide and 13μg/mL catalase (G. Xu et al. 2005 Analytical chemistry 77: 3029-3037).Protein samples are snap frozen on dry ice and stored at −20° C. foranalysis by mass spectrometry.

Standard protocols are used to prepare PD-1 for analysis by MALDI-TOFmass spectroscopy (Example 5). PD-1 is digested with trypsin andMALDI-TOF spectra from 800-5000 m/z are collected and normalized againstthe total peptide present in a sample. Protein Prospector (UCSF) is usedto predict the peptide masses of the MALDI precursor ions. Peakscorresponding to unmodified and oxidized (+16, +32, +48, etc.) states ofeach peptide are visually identified. The fraction of unmodified peptideis calculated from the ratio of the normalized intensity of theunmodified peptide peaks to the sum of the intensities of both theunmodified and oxidized peaks of that peptide. Plotting the fraction ofunmodified peptide against oxidation time or H₂O₂ concentration yieldsdose-response curves that are fit by non-linear regression in GraphPadPrism (M. Brenowitz et al. 2002 Current opinion in structural biology12: 648-653).

Results Fabrication and Characterization of Pyrite Shrink-Film Laminate

Pyrite nanocrystal colloidal suspension is easily sprayed onshape-memory polyolefin using an art supply airbrush (FIG. 1A). A singlepass with the airbrush deposits a 50±25 nm thick coating (FIG. 3C). Twocoats are applied. The sprayed layers are phase-pure pyrite as assayedby powder X-ray diffraction and Raman spectroscopy. The ˜95% areareduction of shrunk shape-memory polyolefin (FIG. 1B) results in a 20fold compression of the planar surface area. Top-down SEMs of laminatesshow wrinkling of the pyrite coating (FIGS. 3, A & B) similar to thewrinkling of metal thin films (D. Nawarathna et al. 2013 Appl Phys Lett102: 63504). Cyclic voltammetry of gold films shows >600% increasedsurface area. We expect similar surface enhancement of the pyrite shrinklaminate due to the similar surface topology (J. D. Pegan et al. 2013Lab on a chip, 13: 4205-4209).

The microwells thermoformed into shrink-film laminate are patterned inthe 384 well format and drawn to a depth sufficient to hold 3 μL (FIGS.1C & D). Thermoforming the sample wells enables the simple samplehandling protocol that we call ‘drop deposition’. Sample handing ismultiplexed with standard multi-channel pipettes.

Pyrite shrink-film laminate has properties that facilitate its use. Thepyrite nanocrystalline coating integrates with the plastic duringshrinkage, stabilizing the surface and thus preventing flaking orchipping. A ‘tape test’ performed on several laminate samples showed novisible transfer of pyrite from the laminates to the tape, demonstratingrobust integration of the pyrite nanocrystals to the polyolefin. While atouch of a pipet tip does not disrupt the laminate surface, pyritenanocrystals deposited on silicon are easily dislodged. Pyriteshrink-film laminate reproducibly generates .OH for >6 months whenstored covered at ambient conditions.

Drop Deposition Oxidation

Optimization of .OH generation as a function of either time or H₂O₂concentration was accomplished by quantifying the oxidation of thefluorescent dye fluorescein. In our protocol, 3 μL drops of samplesolution are deposited, incubated and removed from the microwells (FIG.3). The robust radical production evident for dye incubated withascorbate and H₂O₂ on pyrite shrink-film laminate is enhanced byvibration during sample incubation, presumably by improving the samplingof the solution with the reactive surface (FIG. 3D). The increasedoxidation observed comparing pyrite mineral, pyrite nanocrystalsdeposited on silicon, and pyrite shrink-film laminate shows that boththe nanocrystalline form of pyrite and the wrinkled surface topology ofthe laminate both contribute to .OH production (FIG. 5B).

The production of .OH from pyrite shrink-film laminate was enhanced bysystematically varying the concentrations of sodium ascorbate and H₂O₂.Excessive ascorbate reduces the .OH available to oxidize substratessince it is also a radical scavenger (B. Lipinski, Oxidative medicineand cellular longevity, 2011, 2011, 809696). One mM ascorbate maximizes.OH production as the H₂O₂ concentration is increased from 0 to 2 mM(FIG. 5A). We observe the exponential decay characteristic of a Poissondistribution with pyrite-shrink-film laminate when .OH production iscontrolled by either H₂O₂ concentration or incubation time (FIG. 5C).Achieving ‘single-hit’ distribution of modification or cleavage eventsis advantageous for quantitative footprinting (K. Takamoto and M. R.Chance 2006 Annual review of biophysics and biomolecular structure 35:251-276; M. Brenowitz et al. 1986 Methods in enzymology 130: 132-181)and is conveniently achieved in our standard protocol by varying theH₂O₂ concentration at a constant time of 1 min.

We measured the release of ferrous iron to determine if .OH productionoccurs exclusively on the nanocrystalline surface or also via dissolvedferrous iron. Independent of H₂O₂ concentration, 1.5 μM of iron isleached during the standard 1 min of incubation (FIG. 9). At common H₂O₂and ascorbate concentrations and incubation time, pyrite shrink-filmlaminate produces substantially more oxidation than 2 μM [Fe(edta)]²⁻(FIG. 6), suggesting that the surface is the dominant source of .OH bypyrite shrink-film laminate.

The PD-1 protein retains its native fold following exposure to eithershrink plastic or pyrite shrink-film laminate (Example 10). PD-1 sampleswere incubated on pyrite-shrink-film laminate following our standardprotocol as a function of H₂O₂ concentration and analyzed by MALDI (FIG.3 & Example 5). MALDI is suitable for this study due to the protein'ssmall size; peptides containing >90% of its sequence are detectable(data not shown). The N-terminal peptide (1-36) of PD-1 contains fourresidues susceptible to oxidation and accessible by solvent—Y4, W8, W26,and M31 (FIG. 7B, insert). The +16 ions and multiples thereof areevident as the .OH dose increases as a function of increasing H₂O₂concentration (FIG. 7A). The overall oxidation of this peptide is welldescribed by a Poisson distribution (FIG. 7B). Oxidation was extendedbeyond ‘single-hit’ in this illustration to demonstrate the efficacy of.OH generation (FIG. 7A). An oxidative footprinting analysis wouldutilize lower H₂O₂ concentrations to determine the modification rate andsolvent accessibility and LC-MS/MS to isolate the oxidation of each ofthe susceptible residues.

Discussion

With the advent of protein therapeutics, there is an increased need todevelop high throughput and facile methods to determine proteinstructure to support product development, validation and regulatoryapproval. While atomic resolution models of macromolecular complexes arethe gold standard for structure, oxidative footprinting can contributeto the therapeutic development pipeline by rapidly and inexpensivelymapping the molecular interfaces that mediate a biological activity. Ourgoal in creating pyrite shrink-film laminate is to lower the barrier tooxidative protein footprinting by eliminating the need for a source ofionizing radiation, a UV laser and/or microfluidic sample handling. Ourapparatuses and methods can be implemented using simple, readilyavailable equipment such as a standard laboratory pipette. Higherthroughput can be achieved with a multi-channel pipette or roboticsample handler. Although [Fe(edta)]²⁻ is routinely used in nucleic acidoxidative footprinting (T. D. Tullius and B. A. Dombroski, 1986Proceedings of the National Academy of Sciences of the United States ofAmerica 83: 5469-5473), we have not achieved with this reagent the .OHdose consistency required for protein analysis (unpublished data).Pyrite can also be used to study of nucleic acids and their complexeswith proteins (J. C. Schlatterer et al. 2012 Biochemical and biophysicalresearch communications 425: 374-378).

Pyrite shrink-film laminate is easy and inexpensive to make. While thesynthesis of pyrite nanocrystals requires expertise and an appropriatelyequipped laboratory, it is a straightforward and scalable process.Similarly scalable is the airbrush deposition of pyrite nanocrystals.Other methods for depositing pyrite films can also be used (e.g.,electrodeposition and other low-temperature solution-based approaches).We have prepared prototype shrink-film pieces the size of a standardmicrotiter plate demonstrating a clear path to scaling up to supportrobotic sample handling systems. That pyrite shrink-film laminate can bestably stored will facilitate its adoption.

The laminate device is a promising apparatus for the development of arange of chemical reactions and microfluidic reactors due to bonding ofthe deposited material to the plastic and the enhanced surface area thatresults from shrinkage. Microwell fabrication is completely flexible.The number and volume of the wells is tailored by fabrication of atemplate of any suitable material, such as a PMMA template. Indeed,troughs, serpentine and branched patterns could be molded in thelaminate for use in microfluidic applications. Other materials could bedeposited that would facilitate or catalyze other chemical reactions.Thus, shrink-film laminate is a versatile platform with regard to bothchemistry and physical configuration for the development of novelapplications.

The effectiveness of pyrite shrink-film laminate is enhanced when the.OH dose can be precisely controlled allowing protein oxidation rates tobe calculated. This characteristic, together with a scalable andcustomizable form factor will facilitate higher-throughput analysiswithout the need for infrastructure. We are applying our approach to theanalysis of immunological recognition complexes. Complementarytechniques that backbone solvent accessibility (H/DX 31, proteinpainting; A. Luchini et al. 2014 Nature communications 5: 4413) can beused concomitantly. A challenge to be addressed is the integration ofmultiple structural maps into robust models of molecular structure thatcan be applied to scientific discoveries and industrial processingapplications.

Manufacturers of therapeutic antibodies and innovator and biosimilarbiologics need to map the solvent accessible surface of their moleculesas well as map their target epitopes. Our technology will enable asimple and less expensive way to accomplish these goals for developmentas well as regulatory analysis

Our technology is of great interest to the pharmaceutical industry forthe analysis of biologics as it is simple, easy to use and requires lessmaterial than the other footprinting techniques being applied.

The pyrite shrink-film laminate apparatuses discussed hereinsignificantly reduce the cost of important pharmaceutical analysesincluding protein mapping analysis. For example, it is believed thatthese apparatuses will be advantageous for Fab-antigen and Mab-antigencomplexes.

Example 1 Deposition of Pyrite Nanocrystal Films on Shape-Memory orOther Heat Shrink Polymer

All chemicals were used as received. Anhydrous iron (II) chloride (99.9%metal basis), sulfur powder (99.998%), anhydrous chloroform (≧99%), andanhydrous ethanol (99.5%) were purchased from Sigma Aldrich.Octadecylamine (90%) and diphenyl ether (99%) were purchased from Acros.All synthesis reactions were performed on a Schlenk line under air-freeconditions. Briefly, a solution of 480 mg of sulfur in 10 mL of diphenylether was added to a solution of 368 mg of anhydrous FeCl₂ in 25 g ofoctadecylamine. Both precursor solutions were degassed at 75-85° C. for1 hr and then placed under flowing Ar atmosphere. After heating theoctadecylamine solution to 218° C., the ether solution was injected at210° C. and the reaction stirred at 218° C. for 3 hr to grow thenanocrystals. The reaction was then quenched with a water bath. Once thesolution temperature reached 95° C., 20 mL of ethanol was injected toprevent the octadecylamine from solidifying. The quenched solution wasimmediately centrifuged at 4,400 rpm for 3 min and the resultingprecipitate dispersed in 30 mL of chloroform. The nanocrystals werepurified by two additional rounds of precipitation in ethanol andredispersion in chloroform. A final centrifugation at 3,000 rpm for 2min was performed to remove any aggregates from the nanocrystalsuspension. The resulting colloids (6-8 mg/mL) are stable for up to 2wks when stored in air.

Example 2 Time Frame for Pyrite Shrink-Film Fabrication

The synthesis of the pyrite nanocrystals and the fabrication of pyriteshrink-film laminate take surprisingly little time. A total of eight anda half hours is required to fabricate eight 16-well chips (a total of128 sample wells). The bulk of the time (7′ 45″) is for synthesis of thepyrite nanocrystals. Deposition of the nanocrystals, shrinking thecoated polyolefin and thermoforming the sample wells requires anadditional 45″. It should be noted that fabrication of pyriteshrink-film laminate is amenable to scale up as all of the processes canbe integrated into a roll-to-roll manufacturing line that woulddramatically reduce the fabrication time per piece of pyrite shrink-filmlaminate.

Example 3 Protein Preparation and Characterization

Aliquots of recombinant BirA tagged mouse Programmed Death 1 (PD-1)stored in 20 mM Tris-HCl, pH 8.0+150 mM NaCl were thawed and dialyzedinto standard reaction buffer (20 mM Sodium Cacodylate pH 8.0, 50 mMNaCl, 1 mM EDTA) overnight at 4° C. Intrinsic fluorescence andsedimentation velocity analyses were performed to confirm that theprotein retains its native fold. The intrinsic fluorescence of PD-1 wasmeasured in a 3 mm quartz cuvette in a Fluoromax 3 spectrofluorometer inreaction buffer. Reference spectra for the denatured protein wereobtained in this buffer to which 2 M guanidine was added (P. L.Privalov, Critical reviews in biochemistry and molecular biology, 1990,25, 281-305). Sedimentation velocity analysis of PD-1 was conducted in aBeckman XL-I analytical ultracentrifuge in the Ti-60 rotor at 58,000 rpmand 20° C. using the absorption optics set to 280 nm. Values of thesedimentation and diffusion coefficients were calculated using thetime-derivative method implemented in the program DCDT+ (J. S. Philo,Anal Biochem, 2006, 354, 238-246; W. F. Stafford, 3rd, Anal Biochem,1992, 203, 295-301) and corrected to standard conditions (T. M. Laue etal. The Royal Society of Chemistry, Cambridge, UK, 1992, 90-125). Thefluorescence and sedimentation properties of PD-1 were assayed beforeand after the protein was incubated within microwells drawn into eitherbare shrunk polyolefin or laminate (without H₂O₂ or ascorbate present).

Example 4 Measurement of Oxidation by Fluorescent Dye Degradation

Measurement of the fluorescence loss of an aromatic dye in solution is aconvenient way to assess relative rates of .OH production. This assaywas conducted following published protocols (B. Ou, et al. Journal ofagricultural and food chemistry, 2002, 50, 2772-2777; F. Chen, et al. J.Phys. Chem., 2002, 106, 9485-9490 and I. Shcherbakova et al. Nucleicacids research, 2006, 34, e48). A stock solution of fluorescein(Molecular Probes, F1300) in standard reaction buffer was diluted suchthat the fluorescence of the final solution to be analyzed was withinthe linear dynamic range of a Spectramax M5 plate reader (490 nmexcitation, 520 nm emission and 515 nm emission cutoff).

Example 5 MALDI-TOF Mass Spectral Analysis of Protein Oxidation

One and one half μL of trypsin (sequence grade, Promega) was added tosamples of either oxidized or unoxidized samples of PD-1 in the standardreaction buffer plus the quench solution that had been reduced using98.5 μL of 0.02% proteaseMAX (Promega), 5 mM TCEP (Sigma), 20 mMiodoacetamide (Sigma) in 50 mM ammonium bicarbonate. A 1:20 ratio oftrypsin to protein was added and digestion incubated for 3 hr at 37° C.with shaking. Decreasing the pH below 4 with 10% trifluoroacetic acid(TFA) terminates the reaction. Each digest was divided into aliquots,snap frozen on dry ice, and stored at −20° C. Individual volumes of eachsample were concentrated and de-salted using C18 Zip-tips (Millipore).C18 tips were prepared using 100%, 50%, and 0% acetonitrile in thepresence of 0.1% TFA. The sample aliquots were drawn into the resin tobind the peptides, followed by washing of the resin with 0.1%trifluoroacetic acid to remove salt, and finally eluted onto a MALDIplate using α-cyano-4-cinnamic acid in 70% acetonitrile/0.1% TFA.

Example 6 Measurement of Iron Release from Pyrite Shrink-Film Laminate

To measure Fe²⁺ release from pyrite shrink-film laminate, 3 μL drops ofsolution containing 1 mM ascorbate and H₂O₂ at the indicatedconcentration are pipetted into wells and incubated with vibration for60 s. The sample is collected and stock solutions of sodium acetate,hydroxylamine-HCl and 1,10′-phenanthroline are added to finalconcentrations of 120, 6 and 500 mM, respectively. The final volume of1.2 mL is incubated for 10 min at room temperature. The 508 nmabsorption of the Fe²⁺-1,10′-phenanthroline chelate is compared to acalibration curve constructed from known concentrations of ferrousammonium sulfate hexahydrate (Sigma) (S. A. Kumar et al. Analyticachimica acta, 2014, 851, 87-94).

Example 7 Hydroxyl Radical Generation Using [Fe(Edta)]²⁻

Footprinting using [Fe(edta)]²− was conducted as described in (I.Shcherbakova et al. Nucleic acids research, 2006, 34, e48; E. Heyduk andT. Heyduk, Biochemistry, 1994, 33, 9643-9650). Briefly, dye was dilutedto the desired concentration in the standard reaction buffer and 30 μLwas aliquoted into a microfuge tube. Drops of 0.6 μL of fresh stocksolutions of ascorbate, H₂O₂, and EDTA-(NH₄)₂Fe(SO₄)₂.6H₂O were added tofinal concentrations of 10 mM, 15 mM and 10 μM, respectively. Vortexinginitiated oxidation, which was quenched after 1 min.

Example 8 Pyrite Shrink-Film Laminate Stores Stably for at Least OneYear

Hydroxyl radical (·OH) production from pyrite shrink-film laminatequantified as a function of storage time in a closed container at roomtemperature, in the dark and ambient humidity using the dye degradationassay described above at constant experimental conditions. A 3 μL dropcontaining dye, 8 mM H₂O₂ and 1 mM ascorbate was incubated withvibration for 60 sec and quenched following our standard protocol. Therelative .OH production was normalized to the first measurement taken inOctober of 2013. Pyrite shrink-film laminate clearly does not loseactivity when stored over the 14 months assayed.

Example 9 Ferrous Iron Release from Pyrite Shrink-Film Laminate

Measurement of the amount of iron released from pyrite shrink-filmlaminate during production of .OH. Three μL buffered drops containing 1mM ascorbate, and the indicated concentration of H₂O₂ was incubated onpyrite shrink-film for 1 min with vibration. The released ferrous ironwas assayed by its chelation with 1,10′-phenanthroline as describedabove in this Supplement.

Example 10 The Native Fold of PD-1 is Unaffected by Exposure to EitherBare Shrink-Film or Pyrite Shrink-Film Laminate

Prior to demonstrating protein oxidation, control experiments wereconducted to the PD-1 protein retains its native conformation followingexposure to bare shrink plastic and pyrite shrink-film laminate surface(Supplementary FIG. 1). We verified that the protein is monomeric underour solution conditions by sedimentation equilibrium analysis(Mw=14,967±627 Da). Analytical sedimentation velocity reports the globalconformation of a monodisperse protein; the sedimentation profiles of acontrol PD-1 sample and PD-1 incubated on bare shrink plastic orpyrite-shrink are indistinguishable; denatured protein would displayslower sedimentation and diffusion rates. Intrinsic tryptophanfluorescence is an established measure of the stability of proteinfolds. When tryptophan residues move from a hydrophobic protein coretoward solution, the emission maximum of tryptophan shifts to longerwavelengths. The emission spectra of PD-1 incubated on either materialare indistinguishable from the control spectra that are readilydistinguished from the spectra of the denatured protein. Two independentmeasures confirm that neither the plastic substrate nor pyriteshrink-film laminate surface denature the PD-1 protein.

Some embodiments have been described in connection with the accompanyingdrawings. However, it should be understood that the figures are notdrawn to scale. Distances, angles, etc. are merely illustrative and donot necessarily bear an exact relationship to actual dimensions andlayout of the devices illustrated. Components can be added, removed,and/or rearranged. Further, the disclosure herein of any particularfeature, aspect, method, property, characteristic, quality, attribute,element, or the like in connection with various embodiments can be usedin all other embodiments set forth herein. Additionally, it will berecognized that any methods described herein may be practiced using anydevice suitable for performing the recited steps.

For purposes of this disclosure, certain aspects, advantages, and novelfeatures are described herein. It is to be understood that notnecessarily all such advantages may be achieved in accordance with anyparticular embodiment. Thus, for example, those skilled in the art willrecognize that the disclosure may be embodied or carried out in a mannerthat achieves one advantage or a group of advantages as taught hereinwithout necessarily achieving other advantages as may be taught orsuggested herein.

Although these inventions have been disclosed in the context of certainpreferred embodiments and examples, it will be understood by thoseskilled in the art that the present inventions extend beyond thespecifically disclosed embodiments to other alternative embodimentsand/or uses of the inventions and obvious modifications and equivalentsthereof. In addition, while several variations of the inventions havebeen shown and described in detail, other modifications, which arewithin the scope of these inventions, will be readily apparent to thoseof skill in the art based upon this disclosure. It is also contemplatedthat various combination or sub-combinations of the specific featuresand aspects of the embodiments may be made and still fall within thescope of the inventions. It should be understood that various featuresand aspects of the disclosed embodiments can be combined with orsubstituted for one another in order to form varying modes of thedisclosed inventions. Further, the actions of the disclosed processesand methods may be modified in any manner, including by reorderingactions and/or inserting additional actions and/or deleting actions.Thus, it is intended that the scope of at least some of the presentinventions herein disclosed should not be limited by the particulardisclosed embodiments described above. The limitations in the claims areto be interpreted broadly based on the language employed in the claimsand not limited to the examples described in the present specificationor during the prosecution of the application, which examples are to beconstrued as non-exclusive.

What is claimed is:
 1. A hydroxyl radial generating device, comprising:a substrate layer; and an iron-containing or non-iron containinginorganic or organic layer configured to produce .OH.
 2. The hydroxylradical generating device of claim 1, wherein the substrate layercomprises a material configured to shrink when heated by at least 50%,the pyrite layer configured to shrink when heated by a lesser amountsuch that when the substrate layer and the pyrite layer are shrunken thepyrite layer comprises a textured surface.
 3. The hydroxyl radicalgenerating device of claim 2, wherein the substrate layer and the pyritelayer are in a shrunken configuration.
 4. The hydroxyl radicalgenerating device of claim 1, comprising one or more wells for retaininga liquid sample.
 5. The hydroxyl radical generating device of claim 1,wherein the hydroxyl radical generating device comprises a microtiterplate comprising a plurality of sample wells, each of said wells havingthe pyrite layer therein.
 6. The hydroxyl radical generating device ofclaim 1, wherein the hydroxyl radical generating device comprises amicrofluidic device comprising at least one sample channel and a samplesite in fluid communication with the sample channel, the sample sitehaving the layer of pyrite therein.
 7. The hydroxyl radical generatingdevice of claim 1, wherein the pyrite layer comprises pyrite crystals.8. The hydroxyl radical generating device of claim 1, wherein the pyritelayer comprises nano-scale crystals.
 9. A method for producing ahydroxyl radical generating device, comprising: providing a polymericsubstrate layer; placing a layer of pyrite on a surface of the polymericsubstrate layer to form a multi-layer structure; and applying heat tothe multi-layer structure such that at least the surface of thepolymeric substrate layer contracts; wherein the layer of pyritecontracts to a lesser extent than the surface of the polymeric substratelayer providing a high-surface-area and/or textured surface comprisingthe pyrite layer.
 10. The method of claim 9, wherein the layer of pyritecomprise a layer of pyrite nanocrystals.
 11. The method of claim 9,wherein after placing the layer of pyrite on the surface, the layer ofpyrite comprises a minimum thickness of between 20 and 80 nm.
 12. Themethod of claim 9, further comprising thermoforming a well by heatingthe multi-layered structure above the glass transition temperature ofthe polymeric substrate layer and drawing the multi-layered structureinto a sample site form.
 13. The method of claim 12, wherein the samplesite form comprises a structure comprising a plurality of openings andwherein drawing comprises deforming the multi-layer structure at each ofthe openings into the openings.
 14. The method of claim 9, whereinapplying heat causes a shrinkage of at least about 50% of the area ofthe surface.
 15. The method of claim 9, wherein the layer of pyrite isdeposited by a solution-based deposition method or gas-phase depositionmethod.
 16. A method of analysis, comprising: placing a solutioncomprising a biological substance on a sample site of a hydroxylgenerating device comprising a surface of pyrite; incubating thesolution; and analyzing a sample including proteolytic fragments of thebiological substance.
 17. The method of claim 16, further comprisingperforming drop deposition footprinting by placing the solution in awell of a microtiter plate and thereafter incubating and analyzing thesample.
 18. The method of claim 16, further comprising placing thesample on a sample site of a microfluidic device and thereafterincubating and analyzing the sample.
 19. The method of claim 16, whereinthe surface of pyrite comprises a textured surface comprisingnanocrystals.
 20. The method of claim 16, further comprising combiningan amount of H₂O₂ with the solution prior to incubating the solution.21. The method of claim 16, further comprising incubating the solutionby vibrating the sample test device and the solution.
 22. The method ofclaim 16, further comprising combining the solution with a reactionhalting medium after incubating.
 23. The method of claim 17, furthercomprising activating a surface of a well prior to placing the solutiontherein.
 24. The method of claim 22, wherein the biological substanceincludes DNA, RNA, protein, or any other combination of thesemacromolecules.
 25. A kit for analyzing a sample, comprising: asubstrate comprising a layer of pyrite disposed thereon; and one or morereagents that catalyze the production of hydroxyl radicals from thelayer of pyrite.
 26. The kit of claim 25, wherein the one or morereagents catalyze a Fenton chemical reaction.
 27. The kit of claim 25,wherein the one or more reagents comprise solution(s) of hydrogenperoxide (H₂O₂) and/or ascorbate.
 28. The kit of claim 25, wherein thesubstrate and pyrite layer comprise a portion of a microtiter plate. 29.The kit of claim 25, wherein the substrate and pyrite layer comprise aportion of a microfluidic device.